Cellular regulation of nitric oxide synthase expression and S-nitrosylation

一氧化氮合酶表达和 S-亚硝基化的细胞调节

• 批准号: RGPIN-2014-06583
• 负责人: Choy, Jonathan
• 金额: $ 2.99万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=567906
• 关键词: Cellular; regulation; nitric; oxide; synthase

Nitric oxide (NO) is a bioactive gas that controls a wide array of cell biological processes. It exerts its biological effects mainly by causing targeted protein S-nitrosylation, which is the covalent addition of a nitroso moiety onto target cysteines and is an enzymatically-controlled process in cells. S-nitrosylation controls protein function and has been proposed to be of similar importance to cell biology as protein phosphorylation. However, its regulation and biological effects are relatively understudied. Because of the biological importance of NO, the long-term goal of my research program is to understand the biological effects and cellular regulation of this gas. In order to fully understand the biology of NO, it is important to also study the mechanisms by which its production is regulated. NO is synthesized by enzymes called nitric oxide synthases (NOSs), of which there are three isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). Cytokines modulate the expression of all NOSs, and we have studied the cytokine regulation of iNOS and eNOS expression. With regard to iNOS, we have shown that NO initiates a positive feedback loop that amplifies iNOS expression by inducing Ras S-nitrosylation. We also know that NO-mediated amplification of iNOS expression occurs through increased mRNA translation. However, we do not know how Ras S-nitrosylation is regulated in cells and how iNOS mRNA translation is controlled by the NO-mediated feedback pathway that we have identified. These questions need to be investigated. In addition to iNOS, we have also determined that the cytokine IL-17 up-regulates eNOS expression through an unknown post-translational mechanism. Based on our findings, the short-term objectives of this Discovery Grant are to define the cellular regulation of Ras S-nitrosylation and post-transcriptional regulation of iNOS and eNOS expression. The work proposed will advance our understanding of the cell biological effects of NO and of NOS regulation. The three aims are: Aim 1: Determine the mechanism controlling Ras S-nitrosylation and its effect on Ras function. We have determined that iNOS-derived NO leads to Ras S-nitrosylation but do not know the mechanism by which this occurs. Much of the S-nitrosylation that occurs in cells is enzymatically-regulated by nitrosylases, which catalyze the reaction, and by de-nitrosylases, which reverse it. We will identify the nitrosylases and de-nitrosylases that target Ras. The effect of S-nitrosylation on Ras function will also be determined by examining the effect of this protein modification on the association of Ras with signaling molecules and on Ras localization. Aim 2: Determine the mechanism by which NO amplifies iNOS mRNA translation . We have characterized a new NO-mediated signaling pathway that amplifies iNOS mRNA translation but do not know the specific translational mechanisms involved. We will determine this by identifying iNOS mRNA binding proteins that are affected by NO. The function of candidate proteins will then be determined by inhibiting their expression with siRNA and by mutating their RNA binding motifs in iNOS mRNA sequences. Aim 3: Determine the post-translational mechanism by which IL-17 increases eNOS protein levels. We have shown that IL-17 increases eNOS protein levels through a post-translational process but do not know the mechanism. The effect of IL-17 on eNOS post-translational modifications that are known to affect protein stability, such as phosphorylation and ubiqutinylation, will be determined by proteomic analysis. The role of candidate protein modifications will then be determined by mutating the modification site(s).

一氧化氮（NO）是一种生物活性气体，控制着广泛的细胞生物学过程。它主要通过引起靶蛋白S-亚硝基化来发挥其生物学作用，S-亚硝基化是亚硝基部分共价加成到靶半胱氨酸上，并且是细胞中的酶控过程。S-亚硝基化控制蛋白质的功能，并已被认为对细胞生物学具有与蛋白质磷酸化类似的重要性。然而，其调节和生物学效应相对而言研究不足。由于NO的生物重要性，我的研究计划的长期目标是了解这种气体的生物效应和细胞调节。 为了充分了解NO的生物学，重要的是还要研究其产生的调节机制。NO由称为一氧化氮合酶（NOS）的酶合成，其中有三种同种型：神经元型（nNOS）、诱导型（iNOS）和内皮型（eNOS）。细胞因子调节所有NOS的表达，我们已经研究了细胞因子对iNOS和eNOS表达的调节。关于iNOS，我们已经表明，NO启动一个正反馈回路，通过诱导Ras S-亚硝基化放大iNOS表达。我们还知道，NO介导的iNOS表达的扩增通过增加mRNA翻译发生。然而，我们不知道Ras S-亚硝基化在细胞中是如何调节的，以及iNOS mRNA的翻译是如何由我们已经确定的NO介导的反馈途径控制的。这些问题需要调查。除了iNOS，我们还确定了细胞因子IL-17通过未知的翻译后机制上调eNOS表达。 根据我们的研究结果，本发现补助金的短期目标是确定Ras S-亚硝基化的细胞调控和iNOS和eNOS表达的转录后调控。提出的工作将推进我们的理解NO和NOS调节的细胞生物学效应。目的1：确定Ras S-亚硝基化的控制机制及其对Ras功能的影响。我们已经确定iNOS衍生的NO导致Ras S-亚硝基化，但不知道发生这种情况的机制。细胞中发生的大部分S-亚硝基化反应都受到亚硝酰化酶和去亚硝酰化酶的酶促调节，亚硝酰化酶催化反应，去亚硝酰化酶逆转反应。我们将鉴定靶向Ras的亚硝酰化酶和去亚硝酰化酶。S-亚硝基化对Ras功能的影响也将通过检查这种蛋白质修饰对Ras与信号分子的缔合和对Ras定位的影响来确定。目的2：探讨NO促进iNOS mRNA翻译的机制。我们的特点是一个新的NO介导的信号通路，放大iNOS mRNA的翻译，但不知道具体的翻译机制。我们将确定这一点，通过确定iNOS mRNA结合蛋白，NO的影响。候选蛋白的功能，然后将确定通过抑制它们的表达与siRNA和突变它们的RNA结合基序在iNOS mRNA序列。目的3：探讨IL-17诱导eNOS蛋白表达的翻译后机制。我们已经证明IL-17通过翻译后过程增加eNOS蛋白水平，但不知道其机制。将通过蛋白质组学分析确定IL-17对已知影响蛋白质稳定性的eNOS翻译后修饰（例如磷酸化和泛素化）的影响。然后通过突变修饰位点来确定候选蛋白质修饰的作用。